PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.

[Effect of intestinal nitrate on growth of Klebsiella pneumoniae and its regulatory mechanism].

OBJECTIVE: To explore the effect of intestinal nitrates on the growth of Klebsiella pneumoniae and its regulatory mechanisms. METHODS: K. pneumoniae strains with nitrate reductase narG and narZ single or double gene knockout or with NarXL gene knockout were constructed and observed for both aerobic and anaerobic growth in the presence of KNO3 using an automated bacterial growth analyzer and a spectrophotometer, respectively. The mRNA expressions of narG and narZ in K. pneumoniae in anaerobic cultures in the presence of KNO3 and the effect of the binary regulatory system NarXL on their expresisons were detected using qRT-PCR. Electrophoretic mobility shift assays (EMSA) and MST analysis were performed to explore the specific regulatory mechanisms of NarXL in sensing and utilizing nitrates. Competitive experiments were conducted to examine anaerobic growth advantages of narG and narZ gene knockout strains of K. pneumoniae in the presence of KNO3. RESULTS: The presence of KNO3 in anaerobic conditions, but not in aerobic conditions, promoted bacterial growth more effectively in the wild-type K. pneumoniae strain than in the narXL gene knockout strain. In anaerobic conditions, the narXL gene knockout strain showed significantly lowered mRNA expressions of narG and narZ (P < 0.0001). EMSA and MST experiments demonstrated that the NarXL regulator could directly bind to narG and narZ promoter regions. The wild-type K. pneumoniae strain in anaerobic cultures showed significantly increased expressions of narG and narZ mRNAs in the presence of KNO3 (P < 0.01), and narG gene knockout resulted in significantly attenuated anaerobic growth and competitive growth abilities of K. pneumoniae in the presence of KNO3 (P < 0.01). CONCLUSION: The binary regulatory system NarXL of K. pneumoniae can sense changes in intestinal nitrate concentration and directly regulate the expression of nitrate reductase genes narG and narZ to promote bacterial growth.

Multi-omics profiling reveals dysregulated ribosome biogenesis and impaired cell proliferation following knockout of CDR2L.

BACKGROUND: Cerebellar degeneration-related (CDR) proteins are associated with paraneoplastic cerebellar degeneration (PCD) - a rare, neurodegenerative disease caused by tumour-induced autoimmunity against neural antigens resulting in degeneration of Purkinje neurons in the cerebellum. The pathogenesis of PCD is unknown, in large part due to our limited understanding of the functions of CDR proteins. To this end, we performed an extensive, multi-omics analysis of CDR-knockout cells focusing on the CDR2L protein, to gain a deeper understanding of the properties of the CDR proteins in ovarian cancer. METHODS: Ovarian cancer cell lines lacking either CDR1, CDR2, or CDR2L were analysed using RNA sequencing and mass spectrometry-based proteomics to assess changes to the transcriptome, proteome and secretome in the absence of these proteins. RESULTS: For each knockout cell line, we identified sets of differentially expressed genes and proteins. CDR2L-knockout cells displayed a distinct expression profile compared to CDR1- and CDR2-knockout cells. Knockout of CDR2L caused dysregulation of genes involved in ribosome biogenesis, protein translation, and cell cycle progression, ultimately causing impaired cell proliferation in vitro. Several of these genes showed a concurrent upregulation at the transcript level and downregulation at the protein level. CONCLUSIONS: Our study provides the first integrative multi-omics analysis of the impact of knockout of the CDR genes, providing both new insights into the biological properties of the CDR proteins in ovarian cancer, and a valuable resource for future investigations into the CDR proteins.

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice.

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.

CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.

The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

Cytosolic DNA sensors activation of human astrocytes inhibits herpes simplex virus through IRF1 induction.

Introduction: While astrocytes participate in the CNS innate immunity against herpes simplex virus type 1 (HSV-1) infection, they are the major target for the virus. Therefore, it is of importance to understand the interplay between the astrocyte-mediated immunity and HSV-1 infection. Methods: Both primary human astrocytes and the astrocyte line (U373) were used in this study. RT-qPCR and Western blot assay were used to measure IFNs, the antiviral IFN-stimulated genes (ISGs), IFN regulatory factors (IRFs) and HSV-1 DNA. IRF1 knockout or knockdown was performed with CRISPR/Cas9 and siRNA transfection techniques. Results: Poly(dA:dT) could inhibit HSV-1 replication and induce IFN-ß/IFN-λs production in human astrocytes. Poly(dA:dT) treatment of astrocytes also induced the expression of the antiviral ISGs (Viperin, ISG56 and MxA). Among IRFs members examined, poly(dA:dT) selectively unregulated IRF1 and IRF9, particularly IRF1 in human astrocytes. The inductive effects of poly(dA:dT) on IFNs and ISGs were diminished in the IRF1 knockout cells. In addition, IRF1 knockout attenuated poly(dA:dT)-mediated HSV-1 inhibition in the cells. Conclusion: The DNA sensors activation induces astrocyte intracellular innate immunity against HSV-1. Therefore, targeting the DNA sensors has potential for immune activation-based HSV-1 therapy.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Physicochemical Properties and Antioxidant Activity of CRISPR/Cas9-Edited Tomato SGR1 Knockout (KO) Line.

Tomatoes contain many secondary metabolites such as ß-carotene, lycopene, phenols, flavonoids, and vitamin C, which are responsible for antioxidant activity. SlSGR1 encodes a STAY-GREEN protein that plays a critical role in the regulation of chlorophyll degradation in tomato leaves and fruits. Therefore, the present study was conducted to evaluate the sgr1 null lines based on their physicochemical characteristics, the content of secondary metabolites, and the γ-Aminobutyric acid (GABA) content. The total soluble solids (TSS), titrated acidity (TA), and brix acid ratio (BAR) of the sgr1 null lines were higher than those of the wild type(WT). Additionally, the sgr1 null lines accumulated higher levels of flavor-inducing ascorbic acid and total carotenoids compared to WT. Also, the total phenolic content, total flavonoids, GABA content, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical content of the sgr1 null lines were higher than those of the WT. Therefore, these studies suggest that the knockout of the SGR1 gene by the CRISPR/Cas9 system can improve various functional compounds in tomato fruit, thereby satisfying the antioxidant properties required by consumers.

CXCL8 Knockout: A Key to Resisting Pasteurella multocida Toxin-Induced Cytotoxicity.

Pasteurella multocida, a zoonotic pathogen that produces a 146-kDa modular toxin (PMT), causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. However, its mechanism of cytotoxicity remains unclear. In this study, we expressed PMT, purified it in a prokaryotic expression system, and found that it killed PK15 cells. The host factor CXCL8 was significantly upregulated among the differentially expressed genes in a transcriptome sequencing analysis and qPCR verification. We constructed a CXCL8-knockout cell line with a CRISPR/Cas9 system and found that CXCL8 knockout significantly increased resistance to PMT-induced cell apoptosis. CXCL8 knockout impaired the cleavage efficiency of apoptosis-related proteins, including Caspase3, Caspase8, and PARP1, as demonstrated with Western blot. In conclusion, these findings establish that CXCL8 facilitates PMT-induced PK15 cell death, which involves apoptotic pathways; this observation documents that CXCL8 plays a key role in PMT-induced PK15 cell death.

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia.

Hemifacial microsomia (HFM) is a rare congenital genetic syndrome primarily affecting the first and second pharyngeal arches, leading to defects in the mandible, external ear, and middle ear. The pathogenic genes remain largely unidentified. Whole-exome sequencing (WES) was conducted on 12 HFM probands and their unaffected biological parents. Predictive structural analysis of the target gene was conducted using PSIPRED (v3.3) and SWISS-MODEL, while STRING facilitated protein-to-protein interaction predictions. CRISPR/Cas9 was applied for gene knockout in zebrafish. In situ hybridization (ISH) was employed to examine the spatiotemporal expression of the target gene and neural crest cell (NCC) markers. Immunofluorescence with PH3 and TUNEL assays were used to assess cell proliferation and apoptosis. RNA sequencing was performed on mutant and control embryos, with rescue experiments involving target mRNA injections and specific gene knockouts. CDC27 was identified as a novel candidate gene for HFM, with four nonsynonymous de novo variants detected in three unrelated probands. Structural predictions indicated significant alterations in the secondary and tertiary structures of CDC27. cdc27 knockout in zebrafish resulted in craniofacial malformation, spine deformity, and cardiac edema, mirroring typical HFM phenotypes. Abnormalities in somatic cell apoptosis, reduced NCC proliferation in pharyngeal arches, and chondrocyte differentiation issues were observed in cdc27-/- mutants. cdc27 mRNA injections and cdkn1a or tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia, while sox9a mRNA administration partially restored the defective phenotypes. Our findings suggest a functional link between CDC27 and HFM, primarily through the inhibition of CNCC proliferation and disruption of pharyngeal chondrocyte differentiation.

KCTD Proteins Have Redundant Functions in Controlling Cellular Growth.

We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gß1 was involved in the transcriptional control of gene expression, so we compared the gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 led to numerous changes in the expression levels of other G protein subunit genes, while knockout of KCTD isoforms had the opposite effect, presumably because of their role in regulating levels of Gß1. Our work demonstrates a unique relationship between KCTD proteins and Gß1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

Generation of a homozygous DNAJC19 knockout human embryonic stem cell line by CRISPR/Cas9 system.

The DNAJC19 gene, a member of DNAJ heat shock protein (Hsp40) family, is localized within the inner mitochondrial membrane (IMM) and plays a crucial role in regulating the function and localization of mitochondrial Hsp70 (MtHsp70). Mutations in the DNAJC19 gene cause Dilated Cardiomyopathy with Ataxia Syndrome (DCMA). The precise mechanisms underlying the DCMA phenotype caused by DNAJC19 mutations remain poorly understood, and effective treatment modalities were lacking unitl recently. By using CRISPR-Cas9 gene editing technology, this study generated a DNAJC19-knockout (DNAJC19-KO) human embryonic stem cell line (hESC), which will be a useful tool in studying the pathogenesis of DCMA.

[Prdx1 regulates macrophage polarization by maintaining mitochondrial homeostasis].

In order to investigate the role of Prdx1 in macrophage polarization, mouse leukemia cells of monocyte macrophage (RAW264.7) were treated with lipopolysaccharides (LPS)+ interferon gamma (IFNγ) or IL-4 to induce type 1 macrophage (M1) and type 1 macrophage (M2) macrophages, respectively. The Prdx1 gene knockout cells (Prdx1-/-) were used for the study. Flow cytometry was conducted to detect M1/M2 macrophage markers, and ELISA kits were used to measure M1/M2 cytokine levels. Inducible nitric-oxide synthase (iNOS) activity, arginase-1 (Arg-1) activity, and oxidative damage were also assessed. The Seahorse XFe24 Extracellular Flux Analyzer was employed to measure extracellular acidification rate and oxygen consumption rate. The mitochondrial membrane potential was analyzed using the mitochondrial membrane potential dye (JC-1) fluorescent probe, and mitochondrial superoxide was detected through fluorescence staining. Additionally, the impact of adding a mitochondrial reactive oxygen species (ROS) scavenger on RAW264.7 macrophage polarization was examined. The results demonstrated an increase in ROS, hydrogen peroxide, and 8-hydroxy-2 deoxyguanosine (8-OHDG). Cytotoxicity and mitochondrial toxic effects, including mitochondrial superoxide accumulation, decreased adenosine-triphosphate (ATP) production, reduced mitochondrial membrane potential, and decreased mitochondrial DNA copy number, were observed. Furthermore, down-regulation of translocase of inner mitochondrial membrane 23 (TIM23) mitochondrial protein and mitochondrial stress protein heat shock protein 60 (HSP60) was noted. The extra cellular acidification rate (ECAR) in M1 macrophage polarization in RAW264.7 cells was increased, while oxygen consumption rate (OCR) in M2 macrophages was reduced. These findings indicate that Prdx1 knockout in RAW264.7 cells can inhibit M2 macrophage polarization but promote M1 macrophage polarization by impairing mitochondrial function and reducing oxidative phosphorylation.

CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, Heliocidaris crassispina.

Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the ß4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats.

P2X7 receptors mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow and filtration rate, suggesting that receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. We generated a P2X7 knockout rat on F344 background, hypothesising enhanced renal blood flow and protection from angiotensin-II-induced renal injury. CRISPR/Cas9 introduced an early stop codon into exon 2 of P2rx7, abolishing P2X7 protein in kidney and reducing P2rx7 mRNA abundance by ~ 60% in bone-marrow derived macrophages. The M1 polarisation response to lipopolysaccharide was unaffected but P2X7 receptor knockout suppressed ATP-induced IL-1ß release. In male knockout rats, acetylcholine-induced dilation of the renal artery ex vivo was diminished but not the response to nitroprusside. Renal function in male and female knockout rats was not different from wild-type. Finally, in male rats infused with angiotensin-II for 6 weeks, P2X7 knockout did not reduce albuminuria, tubular injury, renal macrophage accrual, and renal perivascular fibrosis. Contrary to our hypothesis, global P2X7 knockout had no impact on in vivo renal hemodynamics. Our study does not indicate a major role for P2X7 receptor activation in renal vascular injury.

Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.